1. Field of the Invention
The present invention is a continuation-in-part of application Ser. No. 10/007,489, Filed Dec. 5, 2001, incorporated by reference and now U.S. Pat. No. 7,125,982 and related application Ser. No. 10/760,156 (pending) and continued-in-part herein. Thio-phosphate can be used as a feed source substituting for phosphate to chemically modify nucleic acids in vivo. Cells incubated in thio-phosphate containing media incorporate the modified phosphate into the backbone of RNA resulting in RNA modified with nuclease resistant phosphorothioate linkages. The extent of thio-phosphate substitution can be varied by varying the ratio of thio-phosphate to phosphate in the culture media. Cells grown in fully substituted thio-phosphate media generate higher yields of mRNA per cell. The consequences for cells are both profound and subtle allowing for continued growth and viability over several generations.
Reduced ratios of thio-phosphate to phosphate are used to maximize protein synthesis. Both native and recombinant protein synthesis can be significantly enhanced. The optimal ratio of thio-phosphate to phosphate for enhancing protein synthesis varies with the organism as well as the physiological state of the cell. The novel mRNA stabilizing media can be used to increase the protein levels of native and/or recombinant proteins in prokaryotes as well as eukaryotes.
2. Description of Related Disclosures
Phosphorothioate linkages in RNA are known to protect such molecules from degradation by RNases found in serum or inside cells (Matzura and Eckstein (1968) European J. Biochem. 3:448-452). The in vivo incorporation of thio-phosphate into the mRNA backbone provides a ready means to induce the stabilization of cellular mRNAs. Bacterial mRNAs are very unstable with half-lives on the order of ˜5 minutes (Bouvet and Belasco (1992) 360:488-491). Eukaryotic mRNAs are considerably more stable with half-lives that range from 20 minutes to 10 hours or more (Wang, Y. (2002) PNAS 99:5860-5865).
Most methods for increasing the yield of mRNA focus on improved extraction methods that stabilize mRNA in vitro via RNase inhibitors (Lader (2003) U.S. Pat. No. 6,528,641). The present method is quite distinct allowing for both in vivo and in vitro stabilization of mRNA by chemical modification of mRNA. In vivo stabilization of bulk mRNA should be useful for detecting rarer messages, unstable messages, processing intermediates, as well as reducing the quantity of material needed for screening samples for particular mRNAs. Note, the use of fully substituted media does not necessarily preserve the natural state of the cell making it more useful for obtaining qualitative and structural information.
Thio-phosphate can also be used to enhance the protein synthesis of many genes. An advantage of this method is that the compound can be used to induce gene expression. In fact, it is often possible to simply add the modified phosphate to the media or feed source since only a fraction of the total phosphate needs to be modified for enhanced protein expression Thio-phosphate is utilized by a variety of organisms and cell types making it readily adaptable for different applications. Enhanced protein synthesis via thio-phosphate is not restricted by choice of vector or promoter and increases in both recombinant and non-recombinant proteins are obtained. The level of enhancement can be as much as ten fold.
An additional advantage of using chemical modification via thio-phosphate is that many genes can be induced simultaneously resulting in a balanced and coordinated induction of enzymes or proteins. This aspect may be particularly important for eukaryotic cells as protein folding can be a complex event requiring many accessory proteins (Hartl and Hayer-Hartl (2002) Science 295:1852-1858). Modifying proteins important for glycosylation etc. may also be enhanced (Dwek et al (2002) Nature Reviews 1:65-75). Coordinating multiple reaction steps through the increased synthesis of rate limiting enzymes or proteins may also be important for bio-catalytic reactions. For large scale fermentation reactions it is also an advantage that the enhancement is a non-mutable or irreversible event.
Methods to maximize the synthesis of proteins vary and include vectors with strong viral or cellular promoters, gene amplification (Yeung et al (1983) J. Biol. Chem. 258:15179-15185), and viral expression vectors (Kaufman (1990) Methods in Enzymology Vol. 185:155-198). Some attempts have been made to increase the stability of recombinant gene products using stabilizer elements. Hairpins to block the decay of mRNA are often time consuming to generate and not predictable with a real possibility of impairing translation (Arnold et al (1998) RNA 4:319-330; Emory et al (1985) Genes Dev. 6; 135-148). Genetic mutants such as RNase deficient mutants have also been generated. RNase mutants are lethal and partial mutants or temperature sensitive mutants must be obtained (Jain (2002) Mol. Microbiol. 43:1053-1064). The present method provides a powerful means to increase the stability of mRNA without inhibiting mRNA processing or impairing cellular viability. It is a very effective technology protecting mRNA from multiple degradative pathways.